Novel mycobacterium tuberculosis protein composition

ABSTRACT

Mycobacterium tuberculosis  proteins and protein compositions that are components of a desaturase complex are provided. The  Mycobacterium tuberculosis  desaturase complex may include a desaturase and an oxidoreductase. The complex may include the rv3229c and rv3230c gene products of  Mycobacterium tuberculosis . Vectors for expressing the desaturase and the oxidoreductase can be packaged together, including a label that indicates their use as a complex for analyzing desaturation of fatty acids. In addition, methods for screening target ligands specific for a desaturase complex are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This invention claims priority to U.S. Provisional Patent ApplicationSer. No. 60/841,822 filed on Sep. 1, 2006, which is incorporated hereinby reference.

GOVERNMENT INTERESTS

This invention was made with United States government support from theNational Institutes of Health (NIH), grant number GM050853. The UnitedStates government may have certain rights in this invention.

FIELD OF THE INVENTION

This invention is related to the biomedical arts. The present inventionprovides proteins and protein compositions that form a multiproteindesaturase complex, which allows for the characterization of enzymesinvolved in the synthesis of unsaturated fatty acids.

BACKGROUND OF THE INVENTION

The integral membrane desaturases are an enzyme family of immensebiomedical and industrial importance. The significance of thedesaturases arises from their fundamental contributions to lipidcompositions and cellular homeostasis. In both eukaryotes andprokaryotes, desaturases produce essential mono- and polyunsaturatedprecursors to the lipid components of all cell membranes and thus helpto control and maintain membrane function. Therefore, desaturases may beinvolved in human diseases associated with changes in lipid composition,including obesity, diabetes, hypertension, cardiovascular disease,immune disorders, degenerative neurological diseases, and skin diseases.Links between monounsaturated fatty acids and the regulation ofapoptosis, neuronal differentiation, and signal transduction have beenreported. The influence of monounsaturated fatty acids on apoptosis maybe coupled to the development of some tumors (Lu et al., 1997, J. Mol.Carcinog. 20: 204-215; Falvella et al., 2002, Carcinog. 11: 1922-1936).Also, the fatty acid composition of erythrocyte membranes is associatedwith breast cancer risk (Pala et al., 2001, J. Nat Canc. Inst. 93:1088-1095).

Stearoyl-CoA desaturase (SCD) catalyzes the rate-determining step in thesynthesis of monounsaturated fatty acids. SCD introduces a double bondbetween positions 9 and 10 of stearoyl-CoA (18:0) and palmitoyl-CoA(16:0). The activity of SCD influences the fatty acid composition ofmembrane phospholipids, triglycerides, and cholesterol esters.Alterations of SCD activity result in changes of membrane fluidity,lipid metabolism, and metabolic rate.

Transgenic mice (Mus musculus) with a mutation in stearoyl-CoAdesaturase 1 (SCD1) have increased energy expenditure, reduced bodyadiposity, and remain lean when subject to a high calorie diet, despitea higher food intake as compared to control mice (Ntambi et al., 2003,Prog. Lipid. Res. 43: 91-104). These findings, limited to analysis ofthe SCD function, link SCD function to a major health epidemic, obesity,and identify SCD as potential target for anti-obesity drugs.

Unsaturated fatty acids are also precursors of mycolic acid, a wax-likecoating that protects human pathogens such as Mycobacterium tuberculosisfrom desiccation, macrophage attack, water-soluble antibiotics, andother ameliorative agents. Desaturases are of great importance toinsects in the biosynthetic pathways for production of juvenilematuration hormones, and in the use of fatty acids as an energy sourceduring swarming. Desaturases also contribute to the composition of allplant seed oils consumed by humans, and are recognized as relevantenzymes for renewable sources of hydrocarbons.

Each of the above areas involving desaturases has high impact on humanhealth or areas of economic interest. It is therefore important toimprove our understanding of the mechanisms in which fatty aciddesaturation proteins function, and to understand the consequence ofthese enzymatic reactions on cellular structure and function.

The desaturase enzyme family is defined by the Pfam database (Bateman etal., 2004, Nucl. Acids Res. 32: D138-D141). SCD from yeast, rat, andmice are each members of the class III diiron family of enzymes. Thehallmark of the membrane-bound SCD enzymes is that all contain an eighthistidine motif (HX(₃₋₄)H˜ ˜HX(₂₋₃)HH˜ ˜HX(₂₋₃)HH). Site-directedmutagenesis in rat SCD has demonstrated that all eight histidines areessential for activity and it was postulated that at least some of theseresidues were necessary for binding the iron atoms. Four isoforms of SCDhave been identified in mice (SCD1-4). SCD1 is expressed largely in theliver and adipose tissue. SCD2 is expressed in the mouse brain, heart,lungs, kidney, spleen, and adipose tissue. SCD3 is expressed in theskin, Harderian gland, and preputial gland. SCD4 is expressedexclusively in the heart. These mouse isoforms are highly homologous andcontain the histidine motif. The physiological roles of these differentenzyme isoforms are currently not understood.

Saccharomyces cerevisiae (yeast) contains a single, essential gene(OLE1) that codes for a desaturase enzyme that is homologous to mouseSCDs. A yeast mutant lacking the OLE1 gene is incapable of growing inthe absence of unsaturated fatty acids (UFAs). Transformation with anexogenous gene containing desaturase activity would complement an OLE1deficient mutant.

Mycobacterium tuberculosis contains a single essential gene (DesA3) thatcodes for a desaturase enzyme that is also homologous to mouse SCDs.Disruption of the DesA3 gene in Mycobacterium is lethal.

DesA3 has been identified as a possible drug target for the treatment oftuberculosis. The development of efficient drugs that can inhibit ordestroy the activity of this enzyme can only be accomplished uponunderstanding of its function. Currently, little is known about DesA3structure and function. The characterization of DesA3 has been impairedby the lack of understanding of the composition of the enzyme systemrequired for activity. The state of the art involves study of DesA3alone using impure vesicle preparations obtained from mycobacterialhomogenates.

It would be advantageous to identify other essential factors for thefunction of DesA3 and to then develop a system that will enable thedetermination of enhanced levels of DesA3 enzymatic activity in vitro.This knowledge could lead to a better understanding of the physiologicalrole of DesA3 and its isoforms in vivo. The generation of such modelexpression system could also be used for identification of the roles ofthe proteins involved in a multi-protein composition such as desaturase.The present invention addresses these and other related needs.

SUMMARY OF THE INVENTION

The present invention provides isolated proteins that include an aminoacid sequence represented by SEQ ID NO: 9. The isolated proteins arecapable of influencing the desaturation of fatty acids. The isolatedproteins may have at least 90% sequence identity to SEQ ID NO: 9, orthey may have at least 95% sequence identity to SEQ ID NO: 9. Theisolated proteins may include at least 380 amino acids of SEQ ID NO: 9.

The invention further provides an isolated protein composition fromMycobacterium tuberculosis, which includes a desaturase and anoxidoreductase. The protein composition is capable of influencingdesaturation of fatty acids. The desaturase can have at least 95% aminoacid sequence identity to Rv3229c, and the oxidoreductase can have atleast 95% amino acid sequence identity to Rv3230c. Alternatively, thedesaturase can be Rv3229c, and the oxidoreductase can be Rv3230c. Insome embodiments, the isolated protein composition may include bothRv3229c and Rv3230c.

The desaturase can be selected from the group consisting of a fatty aciddesaturase capable of inserting double bonds into fatty acyl chainsderivatized to CoA, glycerols, alkyl ethers, alkenyl ethers,phosphatides, mycolic acids, or glycosidic sugars. Preferably, thedesaturase is Mycobacterium tuberculosis DesA3 (also known as Rv3229c).

The oxidoreductase can be selected from the group consisting ofoxidoreductases that are specific for NADH or NADPH, and that reduceenzyme-bound metal ions including heme groups, iron-sulfur centers andthose bound by amino acid side chains such as histidine, glutamate,aspartate, cysteine, or tyrosine. The oxidoreductase can be cytochromeb5. The oxidoreductase can be cytochrome b5 reductase. Theoxidoreductase can be a flavoprotein. The oxidoreductase can be aniron-sulfur protein. The oxidoreductase may contain properties of bothflavoprotein and iron-sulfur proteins. The oxidoreductase may originatein Gram-positive actinomycetes such as Mycobacterium tuberculosis,Mycobacterium leprae, Mycobacterium bovis, Mycobacterium avis,Mycobacterium smegmatis, and others. Preferably, the oxidoreductase isMycobacterium tuberculosis Rv3230c.

The present invention also relates to methods of screening targetligands specific for a desaturase complex. The methods include preparinga first mixture containing a desaturase and an oxidoreductase byexpressing one or more first genes encoding the desaturase andexpressing one or more second genes encoding the oxidoreductase,contacting the first mixture with a fatty acid to form a second mixture,contacting the second mixture with a target ligand, and determining theactivity of the desaturase. The change in activity of the desaturase iscorrelated with binding of the target ligand to the desaturase complex.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the Mycobacterium tuberculosismulti-protein desaturase complex.

FIG. 2 shows images obtained from a Packard Instant Imager (Packard,Meriden, Conn.) for phosphorescence detection of radioactive decay andquantitative analysis (bottom) of duplicate trials for the conversion of[¹⁴C]-18:0-CoA to [¹⁴C]-18:1-CoA after expression of DesA3 from vectorDesA3HispVV16 in Mycobacterium smegmatis.

DETAILED DESCRIPTION OF THE INVENTION

General overview

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry,immunology, protein kinetics, and mass spectroscopy, which are withinthe skill of art. Such techniques are explained fully in the literature,such as in Sambrook et al., 2000, Molecular Cloning: A LaboratoryManual, third edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor; N.Y.; Sambrook et al., 1989, Molecular Cloning: A LaboratoryManual, second edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; Ausubel et al., 1987-2004, Current Protocols in MolecularBiology, Volumes 1-4, John Wiley & Sons, Inc., New York, N.Y.; Kriegler,1990, Gene Transfer and Expression: A Laboratory Manual, Stockton Press,New York, N.Y.;. Dieffenbach et al., 1995, PCR Primer: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,each of which is incorporated herein by reference in its entirety.Procedures employing commercially available assay kits and reagentstypically are used according to manufacturer-defined protocols unlessotherwise noted.

Generally, the nomenclature and the laboratory procedures in recombinantDNA technology described below are those well known and commonlyemployed in the art. Standard techniques are used for cloning, DNA, RNA,and protein isolation, nucleic acid amplification, and nucleic acid andprotein purification. Generally enzymatic reactions involving DNAligase, DNA polymerase, restriction endonucleases and the like areperformed according to the manufacturer's specifications.

Definitions

“Desaturases” refer to enzymes that remove two hydrogen atoms fromadjacent carbons in an organic compound, creating a carbon/carbon doublebond. Such enzymes can be found in humans and other eukaryotes (such asmonkeys, rats, mice, zebrafish, cows, pigs, sheep, chickens, yeast, andothers), in beneficial microorganisms (such as Streptomyces colieocolor,Streptomyces avermitilis and other bacteria that are responsible for thesynthesis of a wide array of antibiotics), and in pathogens (such asMycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium bovis,Mycobacterium avis, and many other Gram-positive actinomycetes).

“Nucleic acid” or “polynucleotide sequence” refers to a single ordouble-stranded polymer of deoxyribonucleotide or ribonucleotide basesread from the 5′ to the 3′ end. Nucleic acids may also include modifiednucleotides that permit correct read-through by a polymerase and do notalter expression of a polypeptide encoded by that nucleic acid.

“Nucleic acid sequence encoding” refers to a nucleic acid that directsthe expression of a specific protein or peptide. The nucleic acidsequences include both the DNA strand sequence that is transcribed intoRNA, and the RNA sequence that is translated into protein. The nucleicacid sequences include both the full length nucleic acid sequences aswell as non-full length sequences derived from the full lengthsequences. It should be further understood that the sequence includesthe degenerate codons of the native sequence or sequences that may beintroduced to provide codon preference in a specific host cell.

“Coding sequence” or “coding region” refers to a nucleic acid moleculehaving sequence information necessary to produce a gene product, whenthe sequence is expressed.

“Nucleic acid construct” or “DNA construct” refers to a coding sequenceor sequences operably linked to appropriate regulatory sequences so asto enable expression of the coding sequence.

“Isolated,” “purified,” or “biologically pure” refer to material that issubstantially or essentially free from components that normallyaccompany it as found in its native state. Purity and homogeneity aretypically determined using analytical chemistry techniques such aspolyacrylamide gel electrophoresis or high performance liquidchromatography. A protein that is the predominant species present in apreparation is substantially purified. In particular, an isolatednucleic acid of the present invention is separated from open readingframes that flank the desired gene and encode proteins other than thedesired protein. The term “purified” denotes that a nucleic acid orprotein gives rise to essentially one band in an electrophoretic gel.Particularly, it means that the nucleic acid or protein is at least 85%pure, more preferably at least 95% pure, and most preferably at least99% pure.

Two nucleic acid sequences or polypeptides are said to be “identical” ifthe sequence of nucleotides or amino acid residues, respectively, in thetwo sequences is the same when aligned for maximum correspondence asdescribed below. The term “complementary to” is used herein to mean thatthe sequence is complementary to all or a portion of a referencepolynucleotide sequence.

“Percentage of sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison and multiplyingthe result by 100 to yield the percentage of sequence identity.

The term “substantial identity” of polynucleotide sequences means that apolynucleotide comprises a sequence that has at least 25% sequenceidentity. Alternatively, percent identity can be any integer from 25% to100%. More preferred embodiments include at least: 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% compared to a referencesequence using the programs described herein; preferably BLAST usingstandard parameters, as described. One of skill will recognize thatthese values can be appropriately adjusted to determine correspondingidentity of proteins encoded by two nucleotide sequences by taking intoaccount codon degeneracy, amino acid similarity, reading framepositioning and the like.

“Substantial identity” of amino acid sequences for purposes of thisinvention normally means polypeptide sequence identity of at least 40%.Preferred percent identity of polypeptides can be any integer from 40%to 100%. More preferred embodiments include at least 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 98.7%, or 99%. Polypeptides that are“substantially identical” share sequences as noted above except thatresidue positions which are not identical may differ by conservativeamino acid changes. Conservative amino acid substitutions refer to theinterchangeability of residues having similar side chains. For example,a group of amino acids having aliphatic side chains is glycine, alanine,valine, leucine, and isoleucine; a group of amino acids havingaliphatic-hydroxyl side chains is serine and threonine; a group of aminoacids having amide-containing side chains is asparagine and glutamine; agroup of amino acids having aromatic side chains is phenylalanine,tyrosine, and tryptophan; a group of amino acids having basic sidechains is lysine, arginine, and histidine; and a group of amino acidshaving sulfur-containing side chains is cysteine and methionine.Preferred conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine.

A protein “isoform” is a version of a protein with some smalldifferences. For example, the small differences may be a result of asplice variant of the protein, or they may be the result of somepost-translational modification. Often, an isoform of an enzyme may havedifferent catalytic properties than the native form of the enzyme.

A “protein composition”, as used herein, refers to a compositioncomprising two or more proteins mixed together.

A “desaturase system” or “desaturase complex” refers to two or morecomponents that are mixed together in order to facilitate thedesaturation of fatty acids. Such components may include enzymes, e.g.desaturase, cytochrome b5, cytochrome b5 reductase, and others. Thecomponents may further include other proteinaceous or non-proteinaceousmolecules that are involved in desaturation of fatty acids. Thesecomponents may interface together. The components of the desaturasesystem may be structurally or functionally related.

A “target ligand” refers to a ligand that can be identified and/orisolated through a specific binding to one or more components of thedesaturase system. For example, the target ligand may be a protein, anenzyme, an antibody, an enzymatic substrate, or a drug. Binding of thetarget ligand to one or more components of the desaturase system isdependent upon the specificity of the binding site(s) of the one or morecomponents of the desaturase system to the target ligand. Binding of thetarget ligand may influence the activity of the desaturase. The changein activity can be measured, e.g., using a desaturation assay.

An “enzyme assay” or “enzymatic assay” refers to a standard laboratorymethod for measuring enzymatic activity. An enzymatic assay fordetermination of desaturase activity (“desaturation assay”) refers to alaboratory method for determining the activity of the enzyme involved infatty acid desaturation (desaturase).

In one example, the change in 18:1-CoA production can be used fordetermination of the desaturase activity. A method for assay of 18:1-CoAproduction is provided as follows. Radioactive fatty acyl-CoAs wereobtained from American Radiolabeled Chemicals (St. Louis, Mo.). Thereaction mixture contained 20 mM of potassium phosphate and 150˜250 mMof NaCl in a total reaction volume of 200 μL. Aliquots (20 μL) of the M.smegmatis pVV16 control lysate or pVV16-DesA3 total lysate, supernatantor pellet fractions were added in various combinations with aliquots (15μL) of supernatant fraction prepared from either E. coli pQE80 controlsupernatant or pQE80-Rv3230c. The reaction was initiated by addition of0.4 μmol of NADPH, 6 nmol of stearoyl-CoA, 0.03 μCi of[1-¹⁴C]-stearoyl-CoA and 0.2 nmol of FAD in a combined volume of 200 μL.The reaction was incubated at 37° C. for 1 h and stopped by the additionof 200 μL of 2.5 M KOH in ethanol. The mixture was heated at 80° C. for1 h and acidified by the addition of 280 μL of formic acid. Thesaponified fatty acids were extracted with 700 μL of hexane, 200 μL ofthe extract was evaporated to dryness, resuspended in 50 μL of hexaneand separated into saturated and unsaturated acids on a 10%AgNO₃-impregnated thin-layer chromatography plate usingchloroform:methanol:acetic acid:water (90:8:1:0.8) as the developingsolvent. Radioactivity was counted by phosphorimaging using a PackardInstant Imager (Packard, Meriden, Conn.) for 30-60 min. Samples preparedin this manner gave ˜200 total imager units for the major radioactivebands detected, which is within the linear response range of theinstrument. Reactions performed with stearoyl-CoA were also treated bythin-layer chromatography as described above, and the individual bandswere extracted from the plate, methylated and analyzed by GC/MS todetermine fatty acid content.

Desaturases

The present invention provides methods for expression of variousdesaturases as part of the desaturase expression system. The desaturasesmay be eukaryotic (e.g. of human, animal, or plant origin) andprokaryotic (e.g. of bacterial origin). For example, desaturases usefulfor practicing of the invention include soluble desaturases andmembrane-bound desaturases, acyl-lipid desaturases, acyl-coenzyme A(acyl-CoA) desaturases, acyl-acyl carrier protein (ACP) desaturases, andother desaturases. Desaturases can be substrate-specific, which meansthat they can preferably catalyze the oxidation-reduction reactions ofspecific substrates. For example, an oxidoreductase that is specific fora CH—CH group of donors preferably catalyzes oxidation-reductionreactions of substrates containing CH—CH groups to produce alkene bonds.Preferably, the desaturase is a stearoyl-CoA desaturase.

Oxidoreductases

The present invention provides methods for expression of variousoxidoreductases as part of the desaturase expression system.Oxidoreductases are enzymes of EC class 1. Oxidoreductases catalyzeoxidation-reduction reactions, which entail the transfer of electronsfrom a substrate that becomes oxidized (electron donor) to a substratethat becomes reduced (electron acceptor).

The oxidoreductases can be substrate-specific, which means that they canpreferably catalyze the oxidation-reduction reactions of specificsubstrates. For example, an oxidoreductase that is specific for a CH—CHgroup of donors preferably catalyzes oxidation-reduction reactions ofsubstrates containing CH—CH groups. Another form of oxidoreductase maybe specific for transfer of electrons to a protein substrate such asanother oxidoreductase. Preferably, the oxidoreductases for practicingthe invention are cytochrome b5, cytochrome b5-like proteins, andcytochrome b5 reductase, Mycobacterium tuberculosis H37Rv oxidoreductaseRv3230c and related proteins from other prokaryotes.

Vectors

The invention involves genetically engineering a system for theexpression of enzymes involved in fatty acid desaturation. The geneticengineering may include increasing the amount of enzymes involved indesaturation. However, in other instances, the genetic engineering mayadditionally include expression of other non-enzymatic components thatare involved in desaturation.

Engineering of the desaturase expression system involves providing forthe expression of one or more heterologous genes that encode protein(s)involved in desaturation. The heterologous gene may be a gene that isnot naturally present in the desaturase system, or it may be a gene thatis naturally present but is placed in a different genetic context (e.g.,the coding region of the gene is operably linked to a promoter that isnot the gene's natural promoter). Typically, the heterologous gene orthe resulting protein will have one or more properties differing fromthe gene in its natural genetic environment.

One method of expression of proteins of the desaturase system of thisinvention is through the use of vectors such as plasmids, phage,phagemids, viruses, artificial chromosomes and the like. Preferredvectors are expression vectors. Expression vectors contain a promoterthat may be operably linked to a coding region. A gene or coding regionis operably linked to a promoter when transcription of the geneinitiates from the promoter. More than one gene may be operably linkedto a single promoter. In preferred embodiments, at least one desaturasegene and at least one oxidoreductase gene are both operably linked tothe same promoter. In other preferred embodiments, at least onedesaturase gene, at least one cytochrome b5 gene, and at least onecytochrome b5 reductase gene are operably linked to the same promoter.In other preferred embodiments, each of the genes is operably linked toa different promoter. In one aspect, the vector is introduced into anorganism that is suitable for expression of the desaturase system.

A variety of expression vectors may be used for expression in E. coli,insect, yeast, or mammalian cells. Expression vectors that may be usedinclude, but are not limited to, Gateway® Destination vectors(Invitrogen, Carlsbad, Calif.), pQE-30, pQE-40, and pQE-80 series(Qiagen, Valencia, Calif.), pUC19 (Yanisch-Perron et al., 1985, Gene 33:103-119), pBluescript II SK+ (Stratagene, La Jolla, Calif.), the pETsystem (Novagen, Madison, Wis.), pLDR20 (ATCC 87205), pBTrp2, pBTac1,pBTac2 (Boehringer Ingelheim Co., Ingelheim, Germany), pLSA1 (Miyaji etal., 1989, Agric. Biol. Chem. 53: 277-279), pGEL1 (Sekine et al., 1985,Proc. Natl. Acad. Sci. USA. 82: 4306-4310), and pSTV28 (manufactured byTakara Shuzo Co., Shimogyo-ku, Kyoto 600-8688, Japan). When a yeaststrain is used as the host, examples of expression vectors that may beused include pYEST-DES52 (Invitrogen), YEp13 (ATCC 37115), YEp24 (ATCC37051), and YCp50 (ATCC 37419). When insect cells are used as theexpression host, examples of expression vectors that may be used includePfastbac1 (Invitrogen, Carlsbad, Calif.), pVL1393 (BD Biosciences,Franklin Lakes, N.J.) and pIEX (Novagen, Madison, Wis.).

Alternatively, expression kits might be utilized for cell-free proteinexpression. For example, the EasyXpress Protein Synthesis Mini Kit, theEasyXpress Protein Synthesis Mega Kit (Qiagen), the In vitro Director™System (Sigma-Aldrich, St. Louis, Mo.), the TnT Sp6 High-Yield ProteinExpression System (Promega; Madison, Wis.) or the WePro lysate (CellFree Sciences, Yokohama, Japan) might be used. Examples of expressionvectors used for cell-free protein expression include pIX4 (Qiagen;Valencia, Caif.), pFK (Promega, Madison, Wis.) and pEU (Cell FreeSciences, Yokohama, Japan).

Expression of the components of the desaturase system is controlled withthe use of desirable promoters. Essentially any promoter may be used aslong as it can be expressed in the engineered organism. A preferredpromoter for E. coli is the lambda P_(R) promoter. In the presence ofthe product of the lambda C_(l) repressor gene, transcription from thelambda P_(R) promoter may be controlled. At temperatures below 37° C.,the repressor is bound to the lambda P_(R) promoter and transcriptiondoes not occur. At temperatures above 37° C. the repressor is releasedfrom the lambda P_(R) promoter and transcription initiates. Thus, bygrowing the organism containing the vector at 37° C. or above, the genesare expressed.

In one example, a preferred promoter for E. coli is the lac promoter. Inthe presence of allolactose, an alternative product of the metabolism oflactose by beta-galactosidase, transcription from the lac promoter maybe controlled. In the absence of allolactose, the lac repressor is boundto the lac operator and transcription does not occur. In the presence ofallolactose, the repressor is released from the lac operator andtranscription initiates. Thus, by growing the organism containing thevector containing lac operator sequences and lac repressor in thepresence of allolactose, the genes are expressed.

When the organism is a yeast cell, any promoter expressed in the yeaststrain host can be used. Examples include the gal 1 promoter (GAL1),leucine2 promoter (LEU2), tryptophan promoter (TRP), gal 10 promoter,heat shock protein promoter, MF alpha 1 promoter, and CUP 1 promoter.

A ribosome-binding sequence (RBS) (prokaryotes) or an internal ribosomeentry site (IRES) (eukaryotes) may be operably linked to the gene. TheRBS or IRES is operably linked to the gene when it directs propertranslation of the protein encoded by the gene. It is preferred that theRBS or IRES is positioned for optimal translation of the linked codingregion (for example, 6 to 18 bases from the initiation codon). Invectors containing more than one gene, it is preferred that each codingregion is operably linked to an RBS or IRES. A preferred RBS is AGMGGAG.

The gene or genes encoding components of the desaturase complex may alsobe operably linked to a transcription terminator sequence. A preferredterminator sequence is the T7 terminator (pET15b; Novagen, Madison,Wis.).

The coding region of the gene may be altered prior to insertion into orwithin the expression vector. These mutants may include deletions,additions, and/or substitutions. When alterations are made, it ispreferred that the alteration maintains the desired enzymatic functionor specificity of the enzyme. However, in certain embodiments, it may bedesired to alter the specificity of the enzyme. For example, one maywish to alter an oxidoreductase such that the activity of the enzyme ischanged. As another example, altering an oxidoreductase may changephysical properties such as stability or solubility.

When a heterologous gene is to be introduced into an organism that doesnot naturally encode the gene, optimal expression of the gene mayrequire alteration of the codons to better match the codon usage of thehost organism. The codon usage of different organisms is well known inthe art.

The coding region also may be altered to ease the purification orimmobilization. An example of such an alteration is the addition of a“tag” to the protein. Examples of suitable tags include FLAG,polyhistidine, biotin, T7, S-protein, myc-, and GST (Novagen; pETsystem). In a preferred embodiment, the gene is altered to contain ahexo-histidine tag in the N-terminus. The protein may be purified bypassing the protein-containing solution through a Ni²⁺ column.

In other embodiments, the coding regions of two or more enzymes arelinked to create a fusion protein. In preferred embodiments, adesaturase-cytochrome b5 fusion protein is encoded. In another preferredembodiment, the fusion protein comprises a desaturase-cytochrome b5reductase.

In further preferred embodiments, the expression vector of the presentinvention comprises at least one polynucleotide sequence encoding adesaturase and at least one polynucleotide sequence encoding anoxidoreductase. The plasmid may also encode one or more enzymes thatfacilitate fatty acid desaturation.

Expression of a Desaturase System

The optimal function of a multi-protein enzyme complex such asdesaturase requires the presence of all members of the enzyme complex.In one aspect, the invention provides a recombinant expression systemthat includes components of a desaturation complex that are involved infatty acid desaturation. Preferably, these components are enzymes. Atminimum, the desaturation complex includes two enzymes: desaturase andoxidoreductase. In various aspects of the invention, different forms ofdesaturases and oxidoreductases may be used. For example, they may benative (full-length), truncated, mutated, or otherwise modified bymethods known in the art. With respect to the expression system, thedesaturase and oxidoreductase may be homologous, heterologous, or mayconstitute mixtures thereof (i.e., one or more enzymes are homologous,whereas other one or more enzyme are heterologous).

In one aspect, the present invention provides a multi-protein desaturasecomplex from Mycobacterium tuberculosis. The complex includes Rv3229cand Rv3230c gene products. The amino acid sequence of the Rv3230c geneproduct is shown in SEQ ID NO: 9. The complex may also includeadditional gene products.

In one embodiment, individual plasmids may express individual geneproducts that form the desaturase complex. For example, if severalplasmids are used for expression, each plasmid may carry one or moregenes encoding one or more components of a mycobacterial desaturasecomplex. Alternatively, a vector may carry more than one gene encodingmore than one component of a mycobacterial desaturase complex. Thus,expression of multiple proteins of a mycobacterial desaturase complexmay be achieved with the use of only one vector.

In a different aspect, the invention provides multiple vectors thatexpress components of a desaturase complex. In one embodiment,individual plasmids may express individual gene products that form thedesaturase complex. For example, if several plasmids are used forexpression, each plasmid may carry one gene encoding one or morecomponents of a desaturase complex. Alternatively, one vector may carrymore than one gene, each gene encoding one or more components of adesaturase complex. Thus, expression of multiple proteins of adesaturase complex may be achieved with the use of only one vector.

In a preferred embodiment, the genes encoding components of a desaturasecomplex are present in equal proportions. This can be accomplished in avariety of ways, for example by using one vector that has equalproportions of genes from a desaturase complex. These genes may beinserted under the control of same control elements for gene expression.One vector may carry sequences encoding more than one component of thecomplex. In one embodiment, the vector might carry three components ofthe desaturase complex, present in a certain ratio. In a preferredembodiment, the ratio of Rv3230c and Rv3229c coding sequences is 1:1 orthe ratio of SCD, cytochrome b5 and cytochrome b5 reductase codingsequences is 1:1:1. Alternatively, individual genes of the desaturasecomplex may be expressed using separate plasmids. In that case, eachplasmid of the expression system may carry at least one gene encoding acomponent of the complex, while all plasmids have identical controlelements for gene expression.

In a different embodiment, the invention provides a cell-free expressionsystem for desaturases, where genes that encode the desaturase complexare added to the system, proteins are expressed, and enzymaticactivities are determined. The proteins that form the desaturase complexmay be introduced by any method known in the art. Preferably, proteinsthat form the desaturase complex include Rv3229c (mycobacterial DesA3)and Rv3230c (mycobacterial DesA3 oxidoreductase) or SCD, cytochrome b5,and cytochrome b5 reductase. Cell-free expression of one or morecomponents of the desaturase system obviates the need for assembly ofmultiple genes in an expression vector to achieve co-expression.Instead, transcribed mRNA from plasmid can be added to achieve any ratioof translated protein. That is why in some examples it may not benecessary to put multiple genes into expression plasmid backbones.

The desaturase may exist as a separate enzyme or may be a genetic fusionwith an oxidoreductase domain.

The invention also provides an assay system for determination ofdesaturase activity. Preferably, the system includes at least oneexpression vector, preferably a vector that includes genes encoding adesaturase and an oxidoreductase. In a preferred embodiment, the vectorincludes sequences encoding Rv3230c and Rv229c, with the ratio of codingsequences being 1:1. Desaturase activity assays may be conducted withvariants of the components of the expression system. For example,different expression vectors may be used. Also, the individualcomponents that are expressed may be varied. For example, in anotherpreferred embodiment, Rv3229c (mycobacterial DesA3) and Rv3230c(mycobacterial DesA3 oxidoreductase) are combined in a ratio of 10:1. Asanother example, a homolog or a mutant protein may be expressed as apart of the desaturase complex, to study its role in the enzymaticreactions.

In some embodiments, different variants of the genes or proteins areintroduced. These include homologs, mutants, proteins with amino acidssubstitutions, etc., depending on the objective of the investigation.

It is further possible to optimize the cell-free expression system ofthis invention by stabilizing each of the components of the desaturasesystem in its own stabilizing buffer, using methods known in the art.

In a further aspect the present invention includes a desaturase complextogether with at least one co-factor, isolated in its pure form, andthen added to the desaturase complex.

The present invention further provides screening methods that can beused to identify target ligands that bind to a desaturase complex thatis involved in desaturation of fatty acids. The target ligands can befor example be proteins or drugs.

In some aspects, the target ligands can be labeled. The method ofscreening target ligands specific for a desaturase complex includesexpressing one or more genes encoding a desaturase, expressing one ormore genes encoding an oxidoreductase, contacting the expresseddesaturase and oxidoreductase with a fatty acid, providing a targetligand, incubating the target ligand with the expressed desaturase andwith the expressed oxidoreductase, and determining the increase inactivity of 18:1-CoA production, thereby determining whether or not thetarget ligand binds to the desaturase complex. For example, if thetarget ligand is a drug molecule, the target ligand may influence theactivity of the desaturation complex, thereby changing (increasing ordecreasing) fatty acid desaturation, as measured by 18:1-CoA production.One skilled in the art can thus evaluate the efficiency of varioustarget ligands (e.g. drug molecules) for desaturation of fatty acids.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

EXAMPLES

It is to be understood that this invention is not limited to theparticular methodology, protocols, patients, or reagents described, andas such may vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to limit the scope of the present invention, which islimited only by the claims. The following examples are offered toillustrate, but not to limit the claimed invention.

Materials

Total genomic DNA of Mycobacterium tuberculosis H37Rv was obtained fromthe TB Research Materials Facility at Colorado State University (Prof.J. Belisle, Director, NIH NIAD NO1AI75320). This material was used toclone rv3230c and rv3229c (encoding DesA3) by PCR methods.

Total RNA is obtained from mouse liver (SCD1), brain (SCD2), Harderiangland (SCD3), and heart (SCD4) using the TRIzol reagent (Invitrogen).AMV Reverse Transcriptase is from Promega (Madison, Wis.), andAccuPrime™ Pfx DNA polymerase is from Invitrogen (Carlsbad, Calif.).

Gene Cloning

The rv3230c gene was amplified by PCR using the primers5′-cgggGT-ACCatcgagggaaggatgagcaagaaacacacgac-3′ (SEQ ID NO: 1) and5′-cccMG-CTTctagatgtccagcacgcaatcac-3′ (SEQ ID NO: 2). The KpnI andHindIII restriction sites are indicated in capital letters. The PCRcontained 10% dimethyl sulfoxide and consisted of 30 cycles of melt,anneal and extend at temperatures of 94° C., 55° C. and 72° C.,respectively. The resulting DNA fragment was purified by gelelectrophoresis and extracted using a QIAquik Gel Extraction Kit(Qiagen). The PCR product was digested with KpnI and HindIII and ligatedinto similarly digested pQE80 vector to create expression vectorpQE80-Rv3230c. E. coli TOP10 pQE80-Rv3230c transformants were culturedon either Luria-Bertani broth or agar medium, both containing 100 μg/mLof ampicillin. Plasmids isolated from these cells were used to verifythe sequence of the PCR-cloned gene.

The rv3229c gene (encoding DesA3) was amplified using the primers5′-gggaattcCATATGgcgatcactgacgtcgacgtattcgcg-3′ (SEQ ID NO: 3) and5′-ccc-AAGCTTggctgccagatcgtcgggttcgg-3′ (SEQ ID NO: 4). The NdeI andHindIII restriction sites are indicated in capital letters. The PCRcontained 10% dimethyl sulfoxide and consisted of 30 cycles of melt,anneal, and extend at temperatures of 94° C., 55° C., and 72° C.,respectively. The resulting DNA fragments were purified by gelelectrophoresis and extracted using a QIAquik Gel Extraction Kit. ThePCR product was digested with NdeI and HindIII and ligated into thesimilarly digested pVV16 to create expression vector pVV16-DesA3. E.coli TOP10 pVV16-DesA3 transformants were cultured on eitherLuria-Bertani broth or agar medium containing 50 μg/mL of kanamycin.Plasmids isolated from these cells were used to verify the sequence ofthe PCR-cloned gene.

Table 1 shows custom designed primers used to clone SCD genes by athree-step PCR approach. The custom designed primers (Table 1) utilizedin the three reactions are synthesized by and purchased from IntegratedDNA Technologies (Coralville, Iowa). The gene-specific primer (SEQ IDNO: 5) is used in the first PCR reaction to isolate the SCD gene andincorporate a portion of the OLE1 starter sequence. The 2^(nd) Forwardprimer (SEQ ID NO: 6) is used in the second PCR reaction to incorporatethe remainder of the OLE1 starter sequence. The 3^(rd) Forward primer(SEQ ID NO: 7) is used in the third PCR reaction to incorporate aribosomal binding site and the recombination site for Gateway® cloningat the 5′ end of the gene. The Reverse primer (SEQ ID NO: 8) is used inall three PCR reactions to incorporate a recombination site for Gateway®cloning at the 3′ end of the gene. The stop codon is eliminated from theopen reading frame using the Reverse primer such that the 6× His tagfrom the pYES-DEST52 plasmid would be incorporated.

Total RNA is obtained from mouse liver (SCD1), brain (SCD2), Harderiangland (SCD3), and heart (SCD4) using the TRIzol reagent. AMV ReverseTranscriptase is used to generate cDNA from the total RNA. Each plasmidis then used as the template in a three-step PCR. The first and secondPCRs incorporated a 27-amino acid sequence representative of the 27N-terminal codons of the OLE1 gene, which code for the endoplasmicreticulum (ER) localization sequence. Then, a ribosomal binding site andthe recombination sites required for Gateway® cloning are introduced. Byway of the pDONR221 entry clone, Invitrogen's Gateway® cloningtechnology incorporated each gene into the yeast expression plasmidpYES-DEST52. Sequencing of the entire gene is performed to ensure nomutations are introduced during the PCR reactions. The GAL1 promoterinduces the expression of the gene in the presence of galactose and the6× His tag allows for Western blot detection using a His-tag Monoclonalantibody kit available from Novagen.

To create a more stable variant of the SCD protein, one that is noteasily degraded by an ER protease at the N-terminal, a truncated form ofeach mouse SCD isoform is created. PCR primers are designed to eliminateapproximately 30 of the first amino acids corresponding to the proteasesite.

TABLE 1 Custom designed primers utilized in the three-step PCR for SCDcloning SEQ ID Name Sequence NO Primer Info Gene- 5′-CCA AAG GAT GAC TCT5 Forward primer containing specific GCC AGC AGT GGC ATT GTC genespecific overlap region GAC (+ gene specific region)-3′ plus portion ofOLE1 starter sequence. 2^(nd) 5′-CCA ACT TCT GGA ACT 6 Forward primercontaining Forward ACT ATT GAA TTG ATT GAC remainder of OLE1 starter GACCAA TTT CCA AAG GAT sequence. GAC TCT GCC-3′ 3^(rd) 5′-GGGG ACA AGT TTGTAC 7 Forward primer containing Forward AAA GCA GGC TCC AATA ATGribosomal binding site and TCT CCA ACT TCT GGA ACT recombination sitefor ACT ATT G-3′ Gateway ® cloning. Reverse 5′-GGGG AC CAC TTT GTA 8Reverse primer containing CAA GAA AGC TGG GTC (+ recombination site forgene specific region [lacking Gateway ® cloning. stop codon])-3′

Protein Expression

Each SCD expression plasmid is transformed into the yeast strain L8-14C,an OLE1 deficient yeast mutant, according to the Saccharomycescerevisiae EasyComp Transformation Kit (Invitrogen). Transformed cellsare cultured on plates containing minimal medium (0.67% yeast nitrogenbase w/o amino acids; 0.2% casamino acids; 2% BactoTm agar) plus 0.005%histidine, 0.01% leucine, 2% D-glucose, and 0.5 mM UFAs. Cells are thenselected from the plates and streaked on minimal medium platescontaining 0.005% histidine, 0.01% leucine, and 2% D-galactose.Galactose induces expression of the inserted gene by acting on the GALLpromoter region of the pYES-DEST52 plasmid.

Time course expression trials are completed by first inoculating a 10 mlculture (synthetic culture medium without uracil {SC-U} containing 2%glucose and 0.5 mM UFAs) with a single isolated colony. The culture isgrown overnight at 30° C. with agitation set at 280 rpm. After ˜30 hr ofincubation the amount necessary to yield an OD₆₀₀ of 0.4 in a 50 mlculture is transferred to a clean culture tube. The cells are harvestedby centrifugation at 3000×g for 10 minutes, resuspended in a smallvolume of SC-U medium containing 2% galactose (-UFAs), and this solutionis used to inoculate a 50 mL of SC-U medium with 2% galactose. Cells aregrown at 30° C. with agitation set at 280 rpm and samples are taken at0, 4, 8, 12, 16, and 24 hours.

Expression of Rv3230c. E. coli Rosetta2 (Novagen, Madison Wis.)transformed with pQE80-Rv3230c was used for expression studies. Thetransformed strain was cultured on either Terrific Broth orLuria-Bertani agar medium containing 34 μg/mL of chloramphenicol and 100μg/mL of ampicillin. Expression of Rv3230c was induced by addition ofIPTG to 0.5 mM and continued overnight at 16° C. The cells harvestedfrom a 2-L culture (˜5 g) were washed once in 20 mM NaH₂PO₄, pH 7.2,containing 150 mM NaCl and re-suspended in the same buffer at acomposition of 1 g of wet cell paste per 2 mL of buffer. The amino acidsequence of Rv3230c is shown in SEQ ID NO: 9.

Fractionation of Rv3230c. The cell suspension was sonicated for a totalof 150 s using a duty cycle of 15 s on and 45 s off (Fisher Model 550Sonic Dismembrator, ⅛-in horn). During sonication, the temperature ofthe cell suspension was maintained below 10° C. by placing the beaker inan ice water bath containing NaCl. The sonicated cell suspension wascentrifuged at 27,000 g for 30 min and the supernatant fraction wasretained for activity assays.

Cloning of DesA3. The rv3229c gene (encoding DesA3) was amplified usingthe primers 5′-gggaattcCATATGgcgatcactgacgtcgacgtattcgcg-3′ and5′-cccAAGCTTggctgccagatcgtcgggttcgg-3′. The NdeI and HindIII restrictionsites are indicated in capital letters. The PCR contained 10% dimethylsulfoxide and consisted of 30 cycles of melt, anneal, and extend attemperatures of 94° C., 55° C., and 72° C., respectively. The resultingDNA fragments were purified by gel electrophoresis and extracted using aQIAquik Gel Extraction Kit. The PCR product was digested with NdeI andHindIII and ligated into the similarly digested pVV16 to createexpression vector pVV16-DesA3. E. coli TOP10 pVV16-DesA3 transformantswere cultured on either Luria-Bertani broth or agar medium containing 50μg/mL of kanamycin. Plasmids isolated from these cells were used toverify the sequence of the PCR-cloned gene.

Expression of DesA3. Mycobacterium smegmatis (ATCC 700084) wastransformed by electroporation with either pVV16-DesA3 or pVV16containing no insert. These transformants were maintained on eitherMiddlebrook 7H10 agar medium or Middlebrook 7H9 broth enriched withMiddlebrook OADC (oleic acid-albumin-dextrose-catalase, BectonDickinson, Spark Md.) and 20 μg/mL kanamycin. The expression studieswere performed in 2-L of the enriched medium. DesA3 was constitutivelyexpressed at 37° C. The cells were grown for ˜38 h, harvested bycentrifugation and washed once in 20 mM NaH₂PO₄, pH 7.2, containing 500mM NaCl. The amino acid sequence of Rv3229c is shown in SEQ ID NO: 10.

Preparation of M. smegmatis Lysate. The cell paste was re-suspended inthe wash buffer at a composition of 1 g of wet cell paste per 2 mL ofbuffer. The cell suspension was sonicated for a total of 480 s using aduty cycle of 15 s on and 45 s off with the temperature of thesuspension maintained below 10° C. A fraction of the total lysate wasretained for assays and other analyses and the remainder was centrifugedat 27,000×g for 30 min to separate the supernatant and the pelletfractions. The soluble fraction was removed and the pellet fraction wasre-suspended in the same volume as total cell lysate subjected tocentrifugation. The three samples were then treated with the same amountof 50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 1 mMphenylmethysulfonyl fluoride, 1 mM EDTA, 1% Triton X-100, 1% sodiumdeoxycholate, and 0.1% SDS.

For immunoprecipitation, the expressed DesA3 was treated with theprimary mouse anti-His-tagged antibody and analyzed by trypsin digestionand mass spectrometry as described for Rv3230c.

Cloning of all enzymes is designed such that a His (6×)-tag isincorporated to facilitate detection of the expressed protein by Westernand purification by affinity chromatography. To determine the optimaltime for cell harvest after induction, samples are taken at differenttimes and the expression is assessed by Western blot.

Western Blot

The cell lysates were analyzed for total expression and the presence ofRv3230c in the soluble fraction by Western blotting with primary mouseanti-His-tagged antibody (Novagen) and SDS-PAGE. Samples were preparedand fractionated for SDS-PAGE using approaches developed forhigh-throughput structural genomics studies. For theimmunoprecipitation, the lysate was treated with antibody and then theantigen-antibody complex was precipitated by addition of ProteinG-linked Sepharose resin (Amersham, Piscataway, N.J.). The precipitatedsample of Rv3230c was purified by SDS-PAGE and further analyzed bytrypsin digestion and mass spectrometry at the University of WisconsinBiotechnology Center.

In vivo Activity Assay

Full-length and truncated versions of each mouse isoform, SCD1-SCD4 andmycobacterial DesA3, were successfully amplified and cloned. The use ofGateway® technology averaged >95% efficiency. Each SCD isoform istransformed into the OLE1 deficient yeast strain L8-14C. Transformedcells are capable of growing on minimal medium plates containinghistidine, leucine, D-glucose, and UFAs, or on minimal medium platescontaining histidine, and D-galactose, in the absence of unsaturatedfatty acids. The results are summarized in Table 2. These experimentsproved that that the enzymes are active in vivo. Note that the SCD4enzyme appeared to differ from the other isoforms as the transformedyeast exhibited different growth patterns.

TABLE 2 Comparison of expression systems for the mammalian stearoyl-CoAdesaturases (4 mouse (m) isoforms, 2 human (h) isoforms, 1 andmycobacterial (DesA3) isoform Enzyme Protein constructs available Invivo activity mSCD1 Full-length and truncate ++++++ mSCD2 Full-lengthand truncate ++++ mSCD3 Full-length and truncate +++++ mSCD4 Full-lengthand truncate Not active hSCD1 ole 1 chimera +++ hSCD5 Wild-type +++++DesA3 Wild-type and ole 1 chimera None without Rv32320c

TABLE 3 Comparison of reconstituted complexes of recombinant mSCD1(heterologous expression; prepared as yeast microsomes) with exogenousrecombinant cyt b5 and cyt b5 reductase (heterologous expression;prepared in ^(Escherichia coli)) mSCD1 + cyt b5 Complete BackgroundmSCD1 only mSCD1 + cyt b5 reductase complex mSCD1 − − + + + + + + + +cyt b5 − − − − + + − − + + cyt b5 − − − − − − + − + + reductase cpm18:0-CoA 27,392 27,901 28,468 29,673 26,319 21,999 27,015 27,983 25,38021,289 cpm 18:1-CoA 569 562 1626 1743 1684 1761 1372 1513 1599 2018total cpm 37,961 28,463 30,094 31,416 28,003 23,760 28,387 29,496 26,97923,307 % conversion 2.03% 1.97% 5.40% 5.55% 6.01% 7.41% 4.83% 5.13%5.93% 8.66% average 2.00% 5.48% 6.71% 4.98% 7.29% conversion Correctionby 0.00% 3.47% 4.71% 2.98% 5.29% subtraction of background % change35.64%  −14.2%  52.35%  vs. mSCD1

TABLE 4 Comparison of reconstituted complexes of recombinant DesA3(heterologous expression in M. smegmatis) with Rv3230c (heterologousexpression; prepared in Escherichia coli)^(a) M. smegmatis fraction usedTotal Soluble Pellet Total Soluble Pellet Total Soluble Pellet E. colipQE80 − − − + + + − − − control lysate used E. coli pQE80- − − − − −− + + + Rv3230c lysate M. smegmatis pVV16 control lacking DesA3^(c)%18:0 82% 91%  86% 84% 95%  91%  84% 95% 93% %18:1  6% 3%  3%  4% 3% 4% 4%  3%  3% % side product^(d) 12% 6% 11% 12% 2% 5% 12%  2%  4% M.smegmatis pVV16-DesA3^(e) %18:0 74% 91%  74% 77% 94%  83%  66% 83% 63%%18:1 13% 3%  4%  7% 4% 9% 20% 14% 28% % side product 13% 6% 22% 16% 2%8% 14%  3%  9% fold-increase in 2.2 1.0 1.3 1.8 1.3 2.5 5.0 4.7 9.318:1^(f) ^(a)Samples were analyzed by phosphorimaging as describedabove. ^(b)The subcellular fractions of M. smegmatis used in theexperiment were: total cell lysate, soluble fraction or pellet fractionprepared as described above. ^(c)Control studies using M. smegmatispVV16 not able to express DesA3. ^(d)GC/MS analysis of the side productobtained from unlabeled reactions showed this band contained a mixtureof 16:0, 16:1 and 18:0 fatty acids, with 16:1 predominant and 16:0 and18:0 present in trace amounts. Only the 18:0 would be detected by theradiolabeling method. This partial separation of radiolabeled 18:0-CoAfrom total cellular lipids is observed in other studies. ^(e)Desaturasereaction studies using M. smegmatis pVV16-DesA3 expressing DesA3.^(f)The fold increase in 18:1 production relative to the comparablecontrol studies.

The inventors cloned, expressed and isolated both DesA3 (Rv3229c) andRv3230c. The Rv3230c gene is annotated as a putative oxidoreductase inintermediary metabolism. The data presented here demonstrate a functionfor Rv3230c, as a previously unidentified oxidoreductase function, tothe DesA3 complex.

FIG. 2 shows activity assays after expression of DesA3 from vectorDesA3HispVV16 in Mycobacterium smegmatis. The product 18:1-CoA isindicated. T, S, and P correspond to the total, supernatant, and pelletfractions of Mycobacterium smegmatis lysate, respectively. Shown in (A)are data obtained for the control—empty vector pVV16. The upper band isthe unreacted substrate, the bottom band is the side reaction productfrom Mycobacterium smegmatis lysate, and the middle band is the oleicacid product, confirmed by standard reaction. Addition of Rv3230c(expressed and partially purified from Escherichia coli) gives a 10-foldincrease in the rate of production of 18:1-CoA. In different experimentswhere the amount of Rv3230c is varied, the increase in activity ofproduction of 18:1-CoA is up to 30-fold, indicating the existence of amulti-protein composition for DesA3 activity in Mycobacteriumtuberculosis. The decrease in activity beyond addition of the optimalamount of Rv3230c is assigned to excess consumption of the cosubstrateNADPH, which is consistent with unbalanced oxidoreductase activityrelative to desaturase activity.

It is to be understood that this invention is not limited to theparticular devices, methodology, protocols, subjects, or reagentsdescribed, and as such may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention, which is limited only by the claims. Other suitablemodifications and adaptations of a variety of conditions and parameters,obvious to those skilled in the art of genetic engineering, molecularbiology, and biochemistry, are within the scope of this invention. Allpublications, patents, and patent applications cited herein areincorporated by reference in their entirety for all purposes.

1. An isolated protein comprising an amino acid sequence represented bySEQ ID NO: 9, wherein the protein, when combined with a desaturase,increases desaturation of fatty acids.
 2. The isolated protein of claim1, wherein the protein has at least 95% sequence identity to SEQ ID NO:9.
 3. The isolated protein of claim 1, wherein the protein comprises atleast 380 amino acids of SEQ ID NO:
 9. 4. An isolated proteincomposition comprising a desaturase and an oxidoreductase, both fromMycobacterium tuberculosis, wherein the protein composition desaturatesfatty acids.
 5. An isolated protein composition according to claim 4wherein the desaturase has at least 95% amino acid sequence identity toRv3229c.
 6. An isolated protein composition according to claim 4 whereinthe desaturase is Rv3229c.
 7. An isolated protein composition accordingto claim 4 wherein the oxidoreductase has at least 95% amino acidsequence identity to Rv3230c.
 8. An isolated protein compositionaccording to claim 4 wherein the oxidoreductase is Rv3230c.
 9. Anisolated protein composition according to claim 4 wherein the desaturasehas at least 95% amino acid sequence identity to Rv3229c and theoxidoreductase has at least 95% amino acid sequence identity to Rv3230c.10. An isolated protein composition according to claim 4 wherein thedesaturase is Rv3229c and the oxidoreductase is Rv3230c.
 11. A method ofscreening target ligands specific for a desaturase complex, whichcomprises: a) preparing a first mixture containing a desaturase and anoxidoreductase by expressing one or more first genes encoding thedesaturase and expressing one or more second genes encoding theoxidoreductase; b) contacting the first mixture with a fatty acid toform a second mixture; c) contacting the second mixture with a targetligand; and d) determining the activity of the desaturase, wherein thechange in activity of the desaturase is correlated with binding of thetarget ligand to the desaturase complex.
 12. The method of claim 11wherein the desaturase is selected from the group of fatty aciddesaturases capable of inserting double bonds into fatty acyl chainsderivatized to CoA, glycerols, alkyl ethers, alkenyl ethers,phosphatides, mycolic acids, or glycosidic sugars.
 13. The method ofclaim 12 wherein the desaturase is a stearoyl-CoA desaturase.
 14. Themethod of claim 12 wherein the desaturase has at least 95% amino acidsequence identity to Rv3229c.
 15. The method of claim 12 wherein thedesaturase is Rv3229c.
 16. The method of claim 11 wherein theoxidoreductase is selected from the group consisting of oxidoreductasesthat are specific for NADH or NADPH, and that reduce enzyme-bound metalions including heme groups, iron-sulfur centers and those bound by aminoacid side chains such as histidine, glutamate, aspartate, cysteine, ortyrosine.
 17. The method of claim 16 wherein the oxidoreductase is acytochrome b5.
 18. The method of claim 16 wherein the oxidoreductase isa cytochrome b5 reductase.
 19. The method of claim 16 wherein theoxidoreductase has at least 95% amino acid sequence identity to Rv3230c.20. The method of claim 16 wherein the oxidoreductase is Rv3230c.